Since human immuno-deficiency virus (HIV) causing acquired immuno-deficiency syndrome (AIDS) was found, investigations for developing effective anti-HIV drugs have been eagerly undertaken, and recently this field has been remarkably advanced. In the research and development of therapeutic agents for AIDS, other than the research and development of novel chemical agents having an anti-HIV effect, the search for natural substances having anti-HIV effect has been actively made, for example it is reported that compounds having several chemical structures from plants inhibit the replication of HIV-1 or enzymes involved therein (for example, see Che, 1991; Schinazi, 1992; Nasr, Cradock & Johnson, 1992; El-Mekkawy et al., 1995; El-Mekkawy, Meselhy, Kusumoto, Kadota, Hattori, Namba, 1998; Ng, Huang, Fong & Yeung, 1997; Kusumoto & Hattori, 1999).
Physiologically active substances from plants are relatively easily available from plants as raw material. In addition, there are many plants that are used as raw material for Japanese and Chinese medicine or folk remedies around the world, and there are also a great deal of accumulation of information on physiologically active substances. Therefore, the physiologically active substances from plants are expected as effective anti-HIV agents.
However, any physiologically active substances having anti-HIV effect from plants found to date do not have sufficient activity. In addition, as some of the above-mentioned substances from plants have harmful side effects such as toxicity, carcinogenicity and the like, it is difficult to select the most suitable substance as an antiviral agent such as anti-HIV agent or the like. Therefore, it is expected to find the substances having a high antiviral effect (for example, anti-HIV effect) and little harmful side effects, and to develop effective antiviral agents based on the finding for example anti-HIV agents.
The present inventors searched several Egyptian folk medicines with the guidance of anti-HIV effect in the process of search for natural AIDS therapeutic agents, and found that methanol-extraction or water-extraction of Croton tiglium seeds (crude drug name of the seeds: badou (, Crotonis Semen) inhibits HIV proliferation and cytopathogenic effect (CPE) in MT-4 cells in a lower concentration (IC50 is 0.025 μg/mL or 2.0 μg/mL, respectively) than the cytotoxic concentration (selective index is 34.4 or 50.0, respectively) (Kawahata, Otake, Mori, Morimoto, Ueba, Kusumoto et al., 1996). The present inventors particularly found that a phorbol ester of tiglian type contained in Croton tiglium, in particular a specific phorbol derivative has an effect of strongly suppressing the proliferation of AIDS virus.
By the way, in the present anti-HIV therapy, the use of an antiviral agent alone is hardly carried out, and a highly active-retroviral therapy by using simultaneously a plurality of agents is mainly carried out. Behind the trend is the fact that a fully anti-HIV effect cannot be necessarily expected in case where a reverse transcriptase inhibitor, a protease inhibitor or the like usable for AIDS therapy is used alone as an anti-HIV agent.
That is, as the present anti-HIV agents merely suppress or inhibit the replication or maturity of AIDS virus, the AIDS therapy must continuously suppress or inhibit the replication or maturity of AIDS virus. On the other hand, AIDS virus cause mutation for each reverse transcription, and therefore it is apt to acquire any drug resistance, and thus it becomes clear that anti-HIV agents based on the mechanism of action in which the replication or maturity of AIDS virus is suppressed or inhibited is lowered in its clinical effects.
In order to compensate the above-mentioned disadvantages, for example 2-agent combination therapy with two reverse transcriptase inhibitors is performed to suppress the replication of virus. Further, even when the 2-agent combination therapy is applied, in case where AIDS virus resistance to the anti-HIV drug is induced owing to a long-term administration and the therapy does not lead to a sufficient therapeutic effect, a more effective 3-agent combination therapy, that is, for example a 3-agent combination therapy of two reverse transcriptase inhibitors of nucleoside type and one protease inhibitor is applied, and the long-term effect is observed by the clinical test.
As the anti-HIV drugs, currently reverse transcriptase inhibitors and protease inhibitors are commercially available, and integrase inhibitors and co-receptor agents, etc. are being developed.
Under the present state as mentioned above, several inhibitors or antiviral agents, or antidotes against the inhibitors or the antiviral agents are proposed as follows:    Regarding inhibitors: for example JP-A-5-279329 (1993) (HIV protease inhibitors having internal lactam ring), JP-A-5-331067 (1993) (inhibitors of retroviral protease), JP-A-6-25158 (1994) (substituted pyrrolidine derivatives and HIV protease inhibitors), JP-A-6-73004 (1994) (substituted pipecolinuc acid derivatives and HIV protease inhibitors), JP-A-7-285877 (1995) (reverse transcriptase inhibitors of HIV-1), and JP-A-11-322789 (1999) (amino acid derivatives);    Regarding antiviral agents: for example JP-A-5-97888 (1993) (novel oxetanocine derivatives, salts thereof and antiviral agents), JP-A-6-56825 (1994) (benzodiazepines), JP-A-6-234641 (1994) (antiviral combinations), JP-A-6-316524 (1994) (anti-AIDS virus agents), and JP-A-7-82292 (1995) (novel glycyrrhetinic acid related compounds or salts thereof); and    regarding antidotes: for example JP-A-9-30974 (1997) (elimination of toxicity and adverse effect of AIDS virus inhibitor or the like and the production process thereof).
As to the mechanism of action of anti-HIV drugs, it is supposed that there are some active points in the process from the replication to maturity in the infected cells caused by the bonding of HIV on normal host cells (human CD4 positive T lymphocytes or macrophages). Hereinafter, the replication and maturity processes of HIV virus are briefly described based on FIGS. 1 and 2.
FIG. 1 shows a general structure of HIV virus. Virus shell 1 is composed of lipid bilayer 2, and has a structure composed of matrix/protein (MA) with gp41 and gp120 on the surface thereof. In the virus shell 1, core/protein (CA), nucleocapsid/protein (NC), RNA, reverse transcriptase 3, integrase 4 and protease 5, etc. are present.
HIV virus having the structure shown in FIG. 1 is replicated and matured according to the mechanism shown in FIG. 2. In FIG. 2, a virus particle having envelope (virus shell 1) binds to a specific cell membrane molecule (receptor; chemokine and CD4) (process {circle around (1)}). Then, the virus particle is fused to the cell membrane by use of the envelope (process {circle around (2)}), and invades the cell. Uncoated (process {circle around (3)}) virus replicates DNA by reverse-transcribing RNA by its own reverse transcriptase (process {circle around (4)}), the replicated DNA is transported in the cell nucleus (process {circle around (5)}). The DNA in the cell nucleus is integrated with integrase (process {circle around (6)}) to form provirus. Further, the genetic information of HIV is amplified and transcribed, the proteins of HIV are synthesized by the action of protease contained in HIV virus (process {circle around (7)}), the assembly of core proteins (process {circle around (8)}) and the aggregation of envelopes (process {circle around (9)}) are performed to complete the maturity of infectious virus particle (process {circle around (3)}). In addition, unknown active parts or mechanism of action that inhibits virus in the process from binding to maturity is assumed (process {circle around (11)}). Also, the formation, release and maturity of HIV virus by trans Golgi network are performed (processes {circle around (12)}, {circle around (13)} and {circle around (9)}).
The present AIDS therapy is centered on the use of anti-HIV drug composed of two agents: an agent for inhibiting the reverse transcriptase (inhibiting process {circle around (4)}) by which RNA is transcribed to DNA in the process from the binding of HIV virus on cell membrane to the integration in the mechanism shown in FIG. 2, and an agent for inhibiting protease involved in the synthesis of core proteins (inhibiting process {circle around (7)}) in the late process to the maturity of the provirus. And, highly active anti-retroviral therapy in which two or more of these agents are combined is attempted.
On the other hand, the above-mentioned specific phorbol derivatives do not have the above-mentioned representative two anti-HIV effects of the inhibition of reverse transcriptase and the inhibition of protease, and it is expected that the derivatives have another anti-HIV effect, that is, an anti-HIV effect for inhibiting the process from the binding of HIV virus on host cell to the fusion thereto (inhibiting processes {circle around (1)} and {circle around (2)}).
Therefore, it is expected that the use of a combination of a specific phorbol derivative with one or more of the above-mentioned several inhibitors (or the conventional anti-HIV drugs), that is, the use of so-called cocktail products having such a constitution exerts a further effective anti-HIV effect compared with the conventional anti-HIV drugs as the cocktail products act simultaneously on plural processes involved in the replication or maturity of HIV virus.
The present invention is made based on the above-mentioned standpoints, and an object thereof is to provide an antiviral agent (for example anti-HIV agent) having a further high anti-viral effect compared with the prior known antiviral agents, and having little harmful side effects.